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Erythrocyte aging and disease
A tale of membranes and microparticles
S. DINKLA
This chapter is an introduction to the research on
erythrocytes, microparticles and regulatory T cells that
is presented in the subsequent chapters. The first part
of this chapter briefly discusses the main functions
and characteristics of the erythrocyte. The second part
focusses on erythrocyte aging and removal, transfusion
side effects, and anemia of inflammation. The third
part discusses the functions of erythrocyte-derived
and platelet-derived microparticles in the context of
health, disease and transfusion medicine. In the
fourth part, a brief overview of the immune system
and its role in tissue homeostasis is given. In the final
part, the scope of this thesis is presented.
I - The erythrocyte
Erythrocytes, commonly known as red blood cells, are
the most abundant cells in the circulation, and are the
principal means of oxygen delivery to and CO2 removal
from the peripheral tissues. The erythrocyte owes
its ability to transport oxygen to its high hemoglobin
content. In the human lung, the erythrocytes take up
oxygen at the alveolar-capillary interface, which they
release whilst traversing the capillary network in the
peripheral tissues. This oxygen release is stimulated
by the CO2 that is produced by metabolically active
tissues. Upon entering the erythrocyte, CO2 is converted
into HCO3- and H+ by the enzyme carbonic anhydrase.
The subsequent decrease in intracellular pH serves as
a signal for hemoglobin to release oxygen.
Erythrocytes are biconcave disks with an approximate
diameter of 7 µm and maximum thickness of 2.5 µm.
Unlike almost all the other cells of the body,
erythrocytes do not contain intracellular organelles such
as a nucleus, endoplasmic reticulum, Golgi apparatus,
and mitochondria, which enables a maximal hemoglobin
content and plasma membrane deformability.
Erythrocytes must be able to undergo large passive
deformations in order to pass through the narrow
Proefschrift ter verkrijging van de graad van doctor
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Ned Tijdschr Klin Chem Labgeneesk 2016, vol. 41, no. 3
capillaries of the microvasculature and the fenestrae in
the spleen. The shear stress that erythrocytes experience
when traversing the vessels and the deoxygenation of
their hemoglobin trigger the release of potent
vasodilators in the form of S-nitrosothiol and ATP,
which promote the passage of the erythrocyte. ATP
promotes the release of NO by the vascular
endothelium, which is further enhanced by the shear
stress that the passing erythrocytes exert on the
endothelium. This physiological activity is exemplary
for the complex interactions between the erythrocyte
and its environment.
II - Erythrocyte aging and removal
In the red bone marrow, multipotent hematopoietic
stem cells proliferate and differentiate to form
erythroblasts (1), which produce large quantities of
hemoglobin and ultimately expel their nuclei to form
reticulocytes. The reticulocytes mature into erythrocytes
in the bone marrow, a process which is completed
after their release into the circulation. This maturation
process is characterized by loss of intracellular
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Figure 1. Erythrocyte birth, aging and removal.
organelles such as mitochondria and ribosomes (2, 3),
and by extensive membrane remodeling (4, 5). In the
circulation, the average lifespan of the human
erythrocyte is approximately 120 days (6), which
means that 200 billion erythrocytes have to be removed
and replaced every day.
Physiological autoantigens
During their stay in the circulation, erythrocyte aging
results in an increase in autologous IgG binding, which
ultimately leads to the phagocytosis of old erythrocytes
by Kupffer cells in the liver (Figure 1) (7). The work of
Marguerite Kay has uncovered that the binding of
physiological autoantibodies to senescent erythrocytes
is associated with changes in band 3. These changes
are thought to be triggered by the binding of denatured
hemoglobin to the cytoplasmic domain of band 3
(Figure 1) (7). It has been suggested that selective tyrosine
phosphorylation of oxidized band 3 by Syk may play a
role in the recruitment of hemoglobin-bound band 3
molecules in large membrane aggregates that show a
high affinity to physiological autoantibodies (8).
Band 3
The Cl-/HCO3- exchange protein band 3, or anion
exchanger 1, is the most abundant protein in the
erythrocyte membrane, facilitates the transport of CO2
through the body (9), and controls the rate of glycolysis
in the erythrocyte by reversible binding of key enzymes
such as glyceraldehyde 3-phosphate dehydrogenase
(10). Band 3 is present in three distinct protein
complexes within the erythrocyte membrane: as an
ankyrin-bound, tetrameric band 3 complex, as a
dimeric band 3 complex bound to the protein 4.1–GPC
junctional complex, and as freely diffusing dimeric
band 3 complexes (Figure 2) (11). The cytoplasmic
domain of band 3 functions as the main anchorage site
of the plasma membrane for the cytoskeleton by
Figure 2. Band 3 multiprotein complexes in the human erythrocyte membrane. CA II – carbonic anhydrase II, GAPDH – glyceraldehyde
3-phosphate dehydrogenase, GPA – glycophorin A, GPB – glycophorin B, GPC – glycophorin C, Hb – hemoglobin, LW – Lewis blood
group system, PFK – phosphofructokinase, Rh – rhesus blood group system, RhAG – Rh-associated glycoprotein blood group system
(van den Akker et al.(11), adapted from Salomao et al.(17))
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Figure 3. Phospholipid distribution and its regulation in the erythrocyte membrane. ATP – adenosine triphosphate, PC – phosphatidylcholine,
PE – phosphatidylethanolamine, PS – phosphatidylserine, SM – sphingomyelin (© Frans Kuypers 2011)
binding ankyrin, protein 4.1, protein 4.2 and adducin
(12-14), and thereby plays a crucial role in the
mechanical integrity and the deformability of the
erythrocyte. Band 3 also interacts with various integral
membrane proteins including glycophorin A, RhAG
and/or Rh from the rhesus complex (Figure 2) (15, 16).
Erythrocyte deformability
Another mechanism of removal centers on the reduced
deformability of aged erythrocytes (18-20). Erythrocyte
deformability is governed by the viscosity of the
cytoplasm - largely determined by its hemoglobin
concentration -, the surface area-to-volume ratio (S/V),
and the mechanical properties of the plasma membrane
- primarily determined by its protein and lipid
constituent organization - including the interaction
between plasma membrane constituents with the
cytoskeleton (19, 21, 22). As previously mentioned, the
erythrocyte has to undergo large deformations in order
to pass through the narrow capillaries of the
microvasculature and the fenestrae in the spleen.
Poorly deformable erythrocytes tend to get stuck in
the spleen, which may trigger phagocytosis (23-25).
Indeed, enhanced splenic sequestration of abnormal
erythrocytes with reduced deformability is associated
with a decreased life span and anemia in several
erythrocyte membranopathies (26). During its time in
the circulation, the erythrocyte loses membrane
surface due to continuous membrane vesiculation
(27-29). This vesicle shedding is thought to constitute
a mechanism for the removal of damaged/aged
membrane patches, postponing the untimely
recognition and elimination of functional erythrocytes
(29-33). In addition, vesiculation also contributes to
the gradual loss in deformability observed during
physiological erythrocyte aging (34), as it leads to a
reduced S/V ratio and increased mean cellular
hemoglobin concentration (34, 35). This might induce
their eventual removal by splenic sequestration.
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Phosphatidylserine exposure
The lipid part of the plasma membrane of the
erythrocyte is composed of equal proportions of
cholesterol and phospholipids (26). While cholesterol
is evenly distributed between the outer and inner leaflet
of the membrane, the four dominant phospholipids are
asymmetrically distributed. Phosphatidylcholine and
sphingomyelin (SM) are primarily located in the
outer leaflet, while phosphatidylethanolamine and
phosphatidylserine (PS) are predominantly found in the
inner leaflet (Figure 3) (36, 37). Various phospholipid
transport proteins have been implicated in membrane
phospholipid asymmetry. “Flippases” transport
phospholipids from the extracellular to the cytoplasmic
membrane leaflet, while “floppases” do the opposite,
both in an energy-dependent manner (Figure 3).
In contrast, “scramblases” move phospholipids bidirectionally down their concentration gradients by an
energy-independent mechanism that is triggered by a
rise in cytoplasmic calcium (Figure 3) (37). A novel
calcium-activated cation channel (TMEM16F) required
for calcium-induced membrane phospholipid scrambling
was recently identified, and was found to be mutated
in patients with Scott syndrome, a disease characterized
by defective calcium-induced phospholipid scrambling
activity (38-40). The plasma membrane lipid
composition is involved in erythrocyte homeostasis
(41, 42), as exemplified by the altered erythrocyte
morphology and survival in patients with
hemoglobinopathies and severe liver diseases, due to a
disturbed membrane lipid asymmetry and lipid
metabolism, respectively (42, 43). The maintenance of
phospholipid asymmetry, in particular the exclusive
localization of PS in the inner plasma membrane
leaflet, has several functional implications. One such
implication is the association between an increase in
PS exposure and vesiculation (44).
Externalization of PS to the outer leaflet of the plasma
membrane is a hallmark of apoptotic cell death for
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many cell types (45-48). Once exposed, PS is
recognized by macrophages which in turn phagocytoze
the targeted cell, either directly via PS receptors (4951), or through PS-mediated opsonisation of cells with
lactadherin (52, 53). This has led to the broadly
supported postulate that PS exposure by erythrocytes
promotes their removal from the circulation.
PS exposure on erythrocytes indeed leads to their
phagocytosis by Kupffer cells of the liver and
macrophages of the spleen in mice (54). A recent study
has shown that stress-induced PS exposure by
erythrocytes leads to their recognition by PS receptors
TIM-1 and TIM-4, and triggers their removal by
TIM-1 and TIM-4-positive phagocytes (49).
Another study demonstrated that endothelial cells
bind PS-positive erythrocytes via stabillin-1 and 2,
greatly enhancing phagocytosis of these erythrocytes
(55). These findings suggest that liver, splenic, and/or
vascular endothelium cooperate with resident
macrophages in the removal of PS-positive erythrocytes.
Interestingly, few erythrocytes (<1%) in the human
circulation expose PS regardless of their age (56),
suggesting that PS exposure either does not occur during
physiological erythrocyte aging, or leads to the rapid
removal of the PS-exposing erythrocyte. In erythrocyte
pathologies such as sickle cell anemia, thalassemia
and spherocytosis, erythrocyte survival inversely
correlated with phosphatidylserine exposure (57).
Another mechanism involved in erythrocyte removal
involves the membrane protein CD47. CD47 is
generally known as a marker of “self”, as it has been
shown to inhibit erythrocyte phagocytosis by
macrophages of the reticulo-endothelial system (58-60).
A recent study adds another dimension to the CD47
story, by showing that a conformational change in
CD47 induced by experimental erythrocyte aging
enables thrombospondin binding and subsequent
phagocytosis by splenic macrophages in vitro (61). It is
reasonable to assume that several of the abovementioned clearance mechanisms act simultaneously
in an effort to efficiently remove old and/or damaged
erythrocytes in vivo.
Side effects of erythrocyte transfusion
Erythrocyte transfusions are given to raise the
hemoglobin concentration in patients with severe
anemia, or after acute blood loss due to surgery and
trauma. Erythrocytes as well as platelets and plasma
are isolated from whole blood of healthy donors by
centrifugation. The collected erythrocytes are stored
in specially designed plastic bags containing a
preservative solution at 4°C. In the Netherlands, the
leukocyte numbers in the product are strongly reduced
by centrifugation and subsequent filtration prior to
storage in SAG-M preservation solution. Dutch legislation
dictates that erythrocyte concentrates can only be
used for transfusion within 35 days after their
collection, in order to meet the international quality
standards of 75% erythrocyte survival at 24 hours
after transfusion and 0.8% hemolysis in the concentrate
(62, 63).
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Although the more than 100 million annual voluntary
blood donations help save millions of lives worldwide
(64), erythrocyte transfusions can also have serious
side effects, such as acute lung injury, iron deposition
resulting in severe organ damage, vasoconstriction,
and formation of alloantibodies and autoantibodies
(65). There are a number of changes that occur during
erythrocyte storage that may cause the observed side
effects after transfusion. These “storage lesions”
include vesiculation (30, 66), decreased deformability
(67, 68), decreased 2,3-bisphosphoglycerate (69), ATP
and glutathione (70) concentrations, potassium leakage
(71), and hemolysis (72).
Immune-mediated side effects
The erythrocyte contains a complex set of regulatory
systems that may induce erythrocyte removal after
physiological or pathological injury such as osmotic
shock, oxidative stress and/or energy depletion (73).
Modulation of these pathways is progressively lost
during storage (74, 75), and this may result in
accelerated aging and the removal of up to 30% of the
transfused erythrocytes within 24 hours after
transfusion (76). Disruption of these systems likely
include a reduction of the threshold for activation of
the pathways governing PS exposure (77), and may
trigger aberrant expression of pathogenic epitopes on
stored erythrocytes and their vesicles (78).
Frequent erythrocyte transfusions may induce the
formation of alloantibodies. This is especially
problematic in the steadily increasing number of
transfusion-dependent patients. Almost half of these
patients acquire alloantibodies at some point in time,
and in approximately 10% of the patients erythrocyte
autoantibodies are detected. Some patients that
produce these autoantibodies develop autoimmune
hemolytic anemia, which can be life-threatening (79,
80). Observations such as these suggest that erythrocyte
antigenicity changes during storage, potentially
leading to autoantibody production after transfusion
against these neoantigens. Murine studies suggest that
erythrocyte transfusions can augment inflammation
(81, 82), that may enhance the risk of immune
responses towards erythrocyte autoantigens.
Storage duration versus clinical outcome
The wealth of data available on the gradual changes
observed in the erythrocyte during storage (83) has
long since sparked the discussion that longer storage
time increases the risk of transfusion side effects.
Multiple studies have reported that increased storage
age of transfused erythrocytes is an independent risk
factor for a number of adverse endpoints (84-90).
In contrast, several other studies did not find any
difference between short and long stored erythrocytes
on clinical outcome (91-94). The use of either
leukocyte-reduced or non-leukocyte-reduced erythrocyte
products in these studies may explain the observed
discrepancies (95). Thus far, there are no completed
prospective randomized controlled studies examining
the effect of storage duration of transfused erythrocytes
on morbidity and mortality (95-97). To definitively
answer this question two large clinical trials are
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currently underway: The US-based initiative Red Cell
Storage Duration Study (RECESS), and the
complementary UK and Canadian co-initiative Age of
Blood Evaluation (ABLE) (97).
Anemia of inflammation
Inflammation arising from various etiologies,
including autoimmune disorders, infection and in
particular sepsis, promote anemia (98-100). The
preferred treatment is directed at the underlying
disease. However, when inflammation persists there
are only a few options for treatment of “anemia of
inflammation” (98). Approximately 40% of critically
ill individuals, including those with severe sepsis,
receive at least one erythrocyte concentrate in the
intensive care unit, with a mean of five concentrates
per patient, equaling half of the total human blood
volume. In these patients, erythrocyte transfusions are
associated with increased morbidity and mortality
(100). Because anemia is a comorbid condition that is
associated with poor outcomes in various chronic
disease states, understanding its pathogenesis is
essential for the development of new remedies (98).
Next to changes in systemic iron homeostasis and
defective erythropoiesis (98, 99), also a reduced
erythrocyte lifespan contributes to anemia of
inflammation (101-103). While iron homeostasis and
defective erythropoiesis have been extensively studied
in these conditions, erythrocyte survival has received
little attention so far (98). Phagocytic capacity is
enhanced during inflammation (104), providing a
possible explanation for enhanced erythrocyte clearance.
Alternatively, the wide range of changes occurring in
the circulation during systemic inflammation might
impact erythrocyte characteristics directly. Indeed,
alterations in erythrocyte shape, deformability, and
aggregability were observed in patients with severe
sepsis (105, 106). To date no mechanistic explanation
has been provided for these changes.
Erythrocyte membrane lipid remodeling in anemia of
inflammation
Membrane protein modification could not be causally
linked to the altered erythrocyte rheology observed in
patients with severe sepsis (107). However, lipid
metabolism is markedly altered during inflammation,
as exemplified by enhanced lipase activity and by
changes in lipid constituents in the plasma (108), and
could thus affect erythrocyte membrane lipid
composition. This hypothesis is supported by the
finding that the incubation of erythrocytes from
healthy volunteers with plasma of septic patients
resulted in enhanced PS exposure and ceramide
content (109). Ceramide is a bioactive lipid involved in
many cellular processes including apoptosis, senescence
and inflammation (110, 11), possibly associated with
the tendency of signaling receptors to cluster in
ceramide-enriched platforms. In addition, the formation
of these platforms alters membrane curvature and
decreases plasma membrane integrity (112, 113).
Sphingomyelinases (SMases) are the principal enzymes
for the generation of ceramide (114). Since in a variety
of diseases, including sepsis, inflammation triggers
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the secretion of acid SMase in the blood, this SMase is
likely responsible for the ceramide formation in
erythrocytes (109).
III - Microparticles
Microparticles (MPs) were first described by Peter
Wolf in 1967, when he observed a halo of debris
surrounding activated platelets which he termed
“platelet dust” (115). They are defined as plasma
membrane-derived vesicles with a diameter of 100 to
1000 nm that expose molecules specific to the parental
cell (116-118). The majority of the blood-borne MPs
are generated by erythrocytes and platelets (118-120).
Depending on their origin, MPs may contain an array
of signaling molecules, including receptors, cytokines
and bioactive lipids, but also mRNA and microRNA.
This molecular composition renders MPs vectors of
biological information. As such they play an active
role in homeostasis and pathogenesis, the latter
including atherosclerosis, various malignancies,
autoimmune disorders, and infection (117, 121).
Erythrocyte-derived microparticles
During aging, the erythrocyte produces numerous
vesicles, often termed red cell microparticles (RMPs),
that expose PS and autoantigens, which are probably
responsible for their rapid removal from the circulation
(30, 31, 120, 122). Data from in vitro studies suggest
that RMPs are likely to be actively involved in
pathophysiology as well. For example, RMPs from
erythrocyte storage units were found to modulate
platelet function (123), and to be highly procoagulant
(124, 125). RMPs can transfer biologically active
molecules, exemplified by the transfer of CD59 from
control erythrocytes to CD59-lacking erythrocytes of
patients with paroxysmal nocturnal hemoglobinuria
(126). Furthermore, RMPs may contribute to the
procoagulant state and vaso-occlusions in sickle cell
disease (119, 127). RMPs from malaria-infected
erythrocytes were found to be major inducers of systemic
inflammation during malaria infection (128, 129).
Platelet-derived microparticles
In contrast to what the name implies, the majority of
the ‘platelet-derived microparticles’ (PMPs) in the
circulation are not derived from platelets, but from
megakaryocytes (130, 131). PMPs are likely to be
important mediators of coagulation, not only by
exposing procoagulant factors (118, 125, 132), but also
by providing a platform for the binding of additional
platelets to the subendothelial matrix (118, 133). PMPs
may be involved in various other processes, such as
hemostasis, maintenance of vascular health, and
immunity (134). Prominent examples are the
involvement of PMPs in vasoregeneration (135, 136),
and their assistance in leukocyte-leukocyte interaction
via P-selectin binding to PSGL-1 (137). A recent study
showed that enhanced glycoprotein VI-mediated PMP
generation leads to a PMP build-up in the joint fluid of
patients suffering from inflammatory arthritis (but not
osteoarthritis), inducing an inflammatory response via
interleukin-1 signaling (138).
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Microparticles in transfusion medicine
Not only in vivo, but also in vitro during blood bank
storage, erythrocytes and platelets shed MPs (30, 139).
These MPs may be responsible for some of the
side-effects commonly observed after transfusion
(140-144). RMPs from storage products are enriched
in removal signals such as PS, immunoglobulins, and
complement (30, 144). The supernatant of erythrocyte
transfusion units, which contains many RMPs that
were shed during storage, was found to have the ability
to modulate the functions of T cells (141), neutrophils
(145), macrophages (140), and monocytes (146) in
vitro. Transfusion unit supernatants also caused lung
inflammation and coagulopathy in a rat model (147).
These findings strengthen the view that a high dose of
RMPs may induce or augment the inflammatory and/
or immunological side-effects of transfusion, including
autoantibody formation (148).
Next to coagulation, platelets also have an important
role as immune mediators (149, 150), making PMPs
likely candidates for immune regulation as well (143,
151-153). Side-effects of platelet transfusion include
fever and acute lung injury (152), and enhanced PMP
levels in platelet units correlated with various allergic
transfusion reactions (151). The ability of PMPs to
interact with leukocytes (137, 151), as well as their
potential to induce CD40L-mediated B cell activation
(154), underscore their ability to affect the immune
system. The plethora of signaling molecules on PMPs
makes it even more likely that they contribute to the
transfusion burden (134, 155). While most circulating
PMPs originate from megakaryocytes (130, 131),
P-selectin-positive PMPs from platelet transfusion
units constitute an important part of the storage PMP
burden, and likely have distinct biological activities
(151, 156).
IV - The immune system
Our immune system has evolved to defend the body
against harmful pathogens. Immune responses are
strictly regulated to allow rapid initiation of immune
activity upon infection, and quick resolution of the
response after elimination of the pathogen, in order to
prevent tissue damage. In addition, the immune system
must be kept in a resting state in normal situations
to prevent harmful responses against the body’s
own tissues and the commensal microorganisms.
Parallel to immune surveillance, immune cells also
provide essential support for tissue homeostasis and
function (157).
The immune response
Tissue injury caused by pathogens and/or trauma
triggers the local release of various soluble proinflammatory mediators, including chemokines and
the cytokines IL-1 and TNF-α by specialized cells
such as resident macrophages. IL-1 and TNF-α
promote the expression of selectins and integrins by
the local vascular endothelium, enabling the adhesion,
arrest, and extravasation of leukocytes, which
subsequently migrate along the chemokine gradient
towards the inflamed tissue (158). Platelets adhere to
the exposed extracellular matrix and the inflamed
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endothelial cells, further activating the endothelium
and adhering monocytes, and release additional
pro-inflammatory factors including IL-1 (159, 160).
At the site of tissue injury, neutrophils engulf pathogens
and compromised cells, and subsequently release
proteolytic enzymes and oxygen metabolites which
digest the phagocytozed material (158). Monocytes
recruited to the inflamed tissue differentiate into
different types of pro-inflammatory macrophages.
Next to their function in phagocytozing pathogens,
dead cells and cell debris, these macrophages are the
primary orchestrators of tissue inflammation (161).
Within days, a specific response is mustered due to
antigen presentation to T and B cells by antigenpresenting cells like macrophages and dendritic cells.
CD4+ helper T (TH) cells support the further activation
of other effector cells of the adaptive immune system
including B cells and CD8+ cytotoxic T cells, through
cell-cell interactions and the release of cytokines.
The activated and differentiated B cells secrete antibodies
that bind the antigen-exposing pathogen, assisting in
their recognition by other effector cells. Activated
cytotoxic T cells search for infected cells in the body
that expose the antigen they are primed against, and
kill these cells by disrupting their membrane and
releasing an array of cytotoxins (157, 158).
Immune cells regulate tissue homeostasis
Initially, inflamed tissue is dominated by M1-polarized
macrophages which promote further leukocyte influx
and TH1-mediated and TH17-mediated inflammatory
responses by producing IL-1, TNF-α, IL-12 and IL-23.
Inflammation must be resolved in a timely manner to
avoid unnecessary tissue damage. M2-polarized
macrophages that do not secrete IL-12 , but instead
produce anti-inflammatory factors such as IL-10, TGF-β,
IL-1 decoy receptor and IL1-RA, gradually appear in
the inflamed tissue to dampen the inflammatory process
(161, 162). They further consolidate the resolution of
inflammation by inducing regulatory T cell (Treg)
differentiation (163), and by actively recruiting Tregs
through the release of CCL22 (164).
Tregs that express the transcription factor Foxp3 play a
critical role in maintaining immune homeostasis and
dominant self-tolerance through a combination of cellcell interactions, the release of soluble factors including
the anti-inflammatory cytokines IL-10, TGF-β and
IL-35, and the scavenging of the T cell survival factor
IL-2 (Figure 4). The majority of the Foxp3+ Tregs are
produced in the thymus as an antigen-primed and
functionally mature T cell subpopulation (naturallyoccurring Tregs), while some Tregs differentiate from
naïve conventional T cells in the periphery (induced
Tregs) (165, 166). Aside from their ability to suppress
effector cell priming in lymphoid tissues (167-170),
Tregs are also able to suppress myeloid populations
and effector cells in other tissues (171). Tregs have
been shown to drive monocyte differentiation towards
a M2-like macrophage phenotype, suggesting they
consolidate the anti-inflammatory macrophage
phenotype (172).
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Figure 4. Mechanisms potentially used by Tregs to assert tissue homeostasis in the steady state, during 'damage control' in inflamed
tissue, and for infectious tolerance after resolution of inflammation. iTreg – induced Treg cell, TGF-βR – TGF-β receptor, nTreg –
natural Treg cell, CO – carbon monoxide, IFN-γR – IFN-γ receptor (Tang et al. (173))
During the resolution of inflammation, TGF-β and
other growth factors such as VEGF, FGF and PDGF,
that are released by macrophages, platelets (174), Tregs
(171, 175) and various other cell types (174), promote
angiogenesis, tissue regeneration and wound repair.
Macrophages further regulate wound healing by
phagocytozing debris, apoptotic neutrophils, and
extracellular matrix (ECM) components that promote
inflammation (176, 177), and by releasing factors that
control ECM turnover (178). This macrophagegoverned healing process is tightly regulated in order
to restore homeostatic tissue architecture and function,
while preventing fibrosis and scarring.
In summary, the immune system sustains tissue
homeostasis by disposing of invading pathogens and
by supporting tissue repair and maintenance.
V - Scope of the thesis
Physiological erythrocyte aging leads to membrane
alterations ultimately responsible for their removal
from the circulation. Similar membrane changes occur
during erythrocyte blood banking, and could explain
the high removal rate of part of the erythrocytes
shortly after transfusion and the transfusion side
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effects. Furthermore, the changes in the erythrocyte
membrane that occur during systemic inflammation
might explain a reduced erythrocyte lifespan
contributing to the development of anemia. In recent
years, increased awareness of the importance of
microparticles in homeostasis, various diseases and
transfusion medicine, has stimulated research on their
exact nature and function. The aim of this thesis is to
provide more insight into the erythrocyte membrane
changes that occur during storage and systemic
inflammation, including vesiculation. In addition, we
studied the potential impact of platelet-derived
microparticles on the immune system.
The potential formation of pathological antigens
during erythrocyte storage was investigated in Chapter
2. Using patient plasma containing anti-erythrocyte
autoantibodies, immunoprecipitations were performed
with erythrocytes and MPs from concentrates of
increasing storage periods. Antigen recognition by
patient plasma autoantibodies was associated with
storage time, and several membrane proteins could be
identified as candidate antigens. The composition of
the immune complexes recognized on the MPs was
markedly different from those on the erythrocytes,
223
indicating that their immunization potential differs
from that of their parental cells. These data corroborate
the theory that deregulation of the mechanisms
governing erythrocyte aging contributes to transfusioninduced alloantibody and autoantibody formation.
In Chapter 3, we assessed the potential of PS exposure
as a parameter for donor-dependent variation in product
quality. PS exposure was determined just prior to, and
during a 35-day storage period, and was found to
correlate with common blood bank quality parameters
and some donor characteristics. PS exposure increased
with storage time, and correlated with hemolysis and
MP concentration in the concentrate, and with the
plasma hemoglobin concentration of the donor.
Furthermore, the initial level of PS exposure was
found to be predictive for PS exposure after osmotic
stress. These findings support the use of PS exposure
as a donor-dependent, biologically relevant parameter
for erythrocyte transfusion unit quality.
During systemic inflammation, as occurs in patients
with severe sepsis, the lipid metabolism in the
circulation is markedly altered, involving an enhanced
activity of several lipases such as SMases. The functional
consequences of these enzymes on erythrocyte
structure and function were studied in detail in
Chapter 4. We found erythrocytes to be very sensitive to
SMase-induced ceramide formation in the membrane.
Ceramide build-up led to the loss of the discoid shape,
followed by PS exposure and loss of cell integrity.
In this process, markedly enhanced vesiculation
and reduced deformability were also observed.
Erythrocytes aged in vivo and in vitro were more
sensitive to SMase-induced changes than younger
erythrocytes. This study indicates that SMase has the
potential to alter pathophysiologically relevant
erythrocyte parameters, which is especially important
in the context of erythrocyte transfusion in patients
with prolonged systemic inflammation.
Based on the results described in Chapter 4, we
investigated sepsis-associated changes in the erythrocyte
membrane lipid composition. In Chapter 5, we
analyzed the plasma membrane lipid content of freshly
isolated erythrocytes from healthy volunteers after
incubation with the plasma of patients with septic
shock. While ceramide could not be detected, we found
markedly increased levels of lysophosphatidylcholine
(LPC), which is produced by phospholipase A2.
Although secretory phospholipase A2 IIA was greatly
enhanced in the septic patient plasmas, its concentration
did not correlate with the LPC levels. Enhanced LPC
levels were not detected when erythrocytes were
incubated with the plasma of healthy volunteers, in
whom sepsis was simulated using a low dose of
lipopolysaccharide. Interestingly, erythrocyte PS exposure
increased in these subjects after lipopolysaccharide
infusion. These data provide evidence for active
remodeling, during sepsis, of the lipid compartment of
the erythrocyte membrane, which is likely to affect
erythrocyte survival.
During the course of our studies we extensively studied
cell-derived MPs from various sources. The isolation,
analysis and quantification of these MPs is challenging
due to their small and heterogeneous size. Based on
224
our experience, we present our perspective on the do’s
and don’ts regarding RMP and PMP isolation and
characterization in Chapter 6. A novel regulatory
function of PMPs on regulatory T cell stability is
presented in Chapter 7. In this chapter we show
that PMPs selectively bind to a specialized subset of
Foxp3+ regulatory T cells, and that PMPs inhibit
their differentiation into potentially pathogenic
effector cells in a pro-inflammatory microenvironment.
Our data indicate the direct involvement of the
adhesion molecule P-selectin and thus suggest a role
for PMPs in vascular healing by regulating the
regulators at the sites of vascular insult.
In Chapter 8, the findings reported in this manuscript
are summarized and discussed, and future perspectives
are outlined.
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